CN103387502B - Method for extracting L-alanine from L-alanine fermentation liquid - Google Patents
Method for extracting L-alanine from L-alanine fermentation liquid Download PDFInfo
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Abstract
The invention discloses a method for extracting L-alanine from an L-alanine fermentation liquid. The method comprises the following steps: a) after fermentation, obtaining a clear liquid containing the L-alanine through solid-liquid separation of the fermentation liquid; b) adjusting the pH of the clear liquid to 4.5-6.0 with an acid, adjusting the concentration of Fe<2+> ions to 7 mmol/L-18 mmol/L by addition of a soluble Fe<2+> salt, during addition of H2O2 with a volume of 0.15%-0.90% of the volume of the clear liquid, slowly heating to the temperature of 40 DEG C-80 DEG C, and stirring at the temperature of 40 DEG C-80 DEG C; c) adjusting the pH to 7.0-8.0 with an alkali, and obtaining a clear liquid through optionally solid-liquid separation; and d) adding 0.1-0.3% of activated carbon at the temperature of 50 DEG C-80 DEG C, stirring at the temperature of 50 DEG C-80 DEG C, obtaining a discolored liquid through solid-liquid separation for removal of the activated carbon, and then obtaining L-alanine crystals through crystal-growth, filtration, washing and drying after vacuum evaporation concentration. The obtained L-alanine crystals have a light transmission rate of more than 90%.
Description
Technical field
The present invention relates to a kind of method extracting ALANINE from ALANINE fermented liquid.
Background technology
ALANINE is a kind of non-essential amino acid in human body, and it has a lot of application in industries such as medicine, food at present.In pharmaceutical industries, ALANINE is one of main component of a lot of moriamin-s, protein synthesis disorder that ALANINE causes as hepatopathy in treatment, diabetes, acute or chronic renal failure and have positive therapeutic action in the life of the nutrition of maintenance urgent patient, rescue patient, in addition, the important source material of ALANINE or synthesise vitamins B6; In food service industry, ALANINE, as attached dose of a kind of nutrition and a kind of artificial sweetening agent, not only can improve the protein utilization in food and beverage, can also improve the sense of taste of a lot of artificial sweetening agent, make sugariness synergy.The main method that current ALANINE is produced comprise with L-Aspartic acid be substrate enzyme transforming process and take glucose as the anaerobion fermentation method etc. of raw material.Anaerobion fermentation method is because its cheaper starting materials, medium component are simple, low power consumption and other advantages, cheaper compared with enzyme process cost, but because the meta-bolites that can produce other in fermentation process is as pigment, organic acid, macromolecular polysaccharide and albumen etc., therefore comparatively enzyme process is more complicated for its leaching process.The extracting method of currently reported microbial method fermentative production ALANINE adopts embrane method to remove thalline and some macromolecular substance, then after ion exchange resin and activated carbon decolorizing, ALANINE is produced in concentrating under reduced pressure crystallization.Adopt this method to extract ALANINE, step is comparatively loaded down with trivial details, and ion exchange resin needs to carry out manipulation of regeneration with acid, alkali and salt after a procedure, and not only cost is higher, and has certain environmental pollution.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, the present invention adopts a kind of chemical process to carry out desolventing technology to fermented liquid clear liquid, solution after process is again through activated carbon decolorizing, concentrating under reduced pressure post crystallization, ALANINE can be produced, transmittance more than 90%, crystalline mother solution repeatedly after recovery yield can reach more than 94%, more original method operation steps is easier, cost is lower.In addition, the method can also process the fermented feed liquid of metabolic disturbance and the mother liquor repeatedly after condensing crystal, therefrom reclaims and extracts remaining ALANINE, not only increase yield and decrease sewage discharge, more environmental protection.
Therefore the object of the present invention is to provide a kind of method extracting ALANINE from ALANINE fermented liquid, comprise the steps:
A), after fermentation ends, fermentation liquor solid-liquid separation, obtains the clear liquid containing ALANINE;
B) use acid is by clear liquid pH regulator to 4.5-6.0, adds solvable Fe
2+salt makes Fe
2+the final concentration of ion is 7mmol/L-18mmol/L, adds the H of supernatant volume 0.15%-0.90%
2o
2, be slowly warming up to 40 DEG C-80 DEG C in the process, stir at such a temperature;
C) regulate pH to 7.0-8.0 with alkali, then optionally carry out solid-liquid separation and obtain clear liquid;
D) at 50 DEG C-80 DEG C, add the gac of 0.1-0.3%, stir at such a temperature, remove after gac through solid-liquid separation and obtain destainer, after reduction vaporization is concentrated after growing the grain, filtration, washing, drying, obtain ALANINE crystal.
The fermentation process of L-Ala is as bibliographical information (Xueli Zhang Kaemwich Jantama, Moore J C, et.al Production of L-alanine by metabolically engineeredEscherichia coli [J] .Appl Microbiol Biotehnol, 2007,77:355-366. Chinese patent 201110235159.8, a kind of XZ-A26 bacterial strain of L-alanine with high yield and construction process and application), anaerobically fermenting cultivation is carried out to recombination bacillus coli escherichia coli XZ-A26.
According to the present invention one preferred embodiment, step b) described in acid be selected from sulfuric acid.
According to the present invention one preferred embodiment, step a) described in solid-liquid separation process adopt ultrafiltration membrance filter fermented liquid to carry out.The molecular weight cut-off of described ultra-filtration membrane is 50000 dalton to 400000 dalton.
According to the present invention one preferred embodiment, step b) in add solvable Fe
2+salt makes Fe
2+the concentration of ion is 10.8mmol/L.
According to the present invention one preferred embodiment, step b) described in the pH regulator to 5.0 of clear liquid.
According to the present invention one preferred embodiment, step b) described in stir continue at least 2 hours, preferably at least 3 hours.
According to the present invention one preferred embodiment, step b) described in alkali be selected from NaOH.
According to the present invention one preferred embodiment, steps d) described in stir continue at least 1 hour.
According to the present invention one preferred embodiment, step a) described in solid-liquid separation process undertaken by suction filtration.
In order to reduce the loss in leaching process, the washing lotion of single batch of crystalline mother solution and washing crystal can also be added to recovery in the fermented liquid of next batch, through time with after (total cycles of concentration can reach more than 60 times), the average yield of ALANINE can reach 94%.
In order to make leaching process more easy, can by chemical treating process and the coupling of activated carbon decolorizing process, such as, at H
2o
2process ferment filtrate, after 3 hours, regulates pH to 7.0-8.0 with alkali lye, directly adds gac, and be slowly warming up to 80 DEG C, and holding temperature and stirring after 1 hour, then obtain the clear liquid that decolours after solid-liquid separation.Clear liquid stirs growing the grain after reduction vaporization is concentrated, and after filtration, washing, drying, can obtain white ALANINE crystal, transmittance is greater than 90%.
In ALANINE fermenting process, likely fermentating metabolism can be caused abnormal because of contaminating into other miscellaneous bacteria and the reason such as fermentation processes is improper, show as ALANINE output in fermented liquid to reduce, and along with the change of fermented liquid character, as color burn, viscosity become large, ion increases, produces the phenomenons such as other small molecular organic acids, finally cause the ALANINE crystal color that extracts excessively dark, transmittance is lower.In addition, mother liquor after the fermentation liquor of Metabolism of Normal is repeatedly concentrated, due to the enrichment of impurity, and in heating concentration process macromolecular substance there is complicated chemical reaction and cause darkening, viscosity improves, clarity step-down, be difficult to continue recycling, can only emit at present.The mother liquor of the inventive method to the fermented feed liquid of above-mentioned metabolic disturbance and repeatedly condensing crystal also has good treatment effect, still can obtain by concentrating under reduced pressure crystallization the ALANINE product that transmittance is greater than 90% by the feed liquid after present method process.
Mother liquor repeatedly after condensing crystal, after dilute with water 1 ~ 3 times, operates by the inventive method, and can obtain white ALANINE crystal, transmittance is greater than 90%.Yield can reach more than 50%.
The ALANINE transmittance detection method obtained by the inventive method: get product 1.0g, after the 20ml that adds water dissolves, measures transmittance with ultraviolet-visible spectrophotometer in 430nm wavelength place.
Accompanying drawing explanation
Fig. 1 is the schema of the inventive method.
Embodiment
The preparation of fermented liquid: the preparation method of fermented liquid is as the method Xueli ZhangKaemwich Jantama of bibliographical information, Moore J C, et.al Production of L-alanine bymetabolically engineered Eschierichia coli [J] .Appl Microbiol Biotehnol, 2007,77:355-366;
And Chinese patent 201110235159.8, a kind of XZ-A26 bacterial strain of L-alanine with high yield and construction process and application.Concrete grammar is as follows:
Bacterial classification: XZ-A26 culture presevation number (CGMCC No.4036)
Seed culture medium and fermention medium form: glucose 120g/L, ammonium chloride 5g/L, NaH
2pO
45g/L, Na
2hPO
45g/L, MgSO
47H
2o 1g/L, CaCl
222H
2o 0.1g/L, small-scale inorganic salt 5ml/L, medium pH 6.5.
Small-scale inorganic salt forms: FeCl
36H
2o 1.5mg, CoCl
26H
2o 0.1mg, CuCl
22H
2o0.1mg, ZnCl
20.1mg, Na
2moO
42H
2o 0.1mg, MnCl
24H
2o 20.2mg, distilled water is settled to 1L, filtration sterilization.
Configure seed culture medium with triangular flask, after 121 DEG C of sterilizing 15min, access XZ-A26, cultivates 18 hours at 30 DEG C.
15L fermentation tank culture medium volume 12L, after 121 DEG C of sterilizing 15min, inoculum size 0.1% (V: V), leavening temperature 30 DEG C, mixing speed 100rpm.With ammoniacal liquor control pH 6.5, fermentation time 48 hours.
Embodiment 1
Fermented liquid 1.5L, ALANINE concentration 78.7mg/ml, obtain fermented liquid clear liquid after crossing 400000 molecular weight ultra-filtration membranes, use H
2sO
4regulate clear liquid pH to 5.0, add FeSO
47H
2o 4.5g, after stirring and dissolving, maintains and stirs, drip the H of 30%
2o
210ml, and be slowly warming up to 50 DEG C, holding temperature and stirring 3h, regulate pH to 7.0 by NaOH solution, use diatomite drainage, suction filtration reaction solution is also washed with water top, obtains clear liquid; Clear liquid adds gac 5g, stirs and is warming up to 80 DEG C, maintains 1h, suction filtration is removed gac and is obtained the clear liquid that decolours, and clear liquid, at 70 DEG C, is evaporated to 70ml, after concentrated solution stirred crystallization 8h, suction filtration obtains crystal, with deionized water wash 2 times, and dry 1h at 80 DEG C, obtain 100.3g ALANINE crystal, purity 99.3%, crystalline mother solution and crystal washing lotion merge 135ml altogether, and ALANINE concentration is 133mg/ml.Yield 84.4%.Transmittance 95.9%.
Embodiment 2
Fermented liquid 1.5L, ALANINE concentration 78.7mg/ml, add crystalline mother solution and the crystal washing lotion amalgamation liquid of embodiment 1, obtains fermented liquid clear liquid, use H after crossing 100000 molecular weight ultra-filtration membranes
2sO
4regulate clear liquid pH to 5.0, add FeSO
47H
2o 4.5g, after stirring and dissolving, maintains and stirs, drip the H of 30%
2o
215ml, and be slowly warming up to 50 DEG C, holding temperature and stirring 3h, regulate pH to 7.0 by NaOH solution, use diatomite drainage, suction filtration reaction solution is also washed with water top, obtains clear liquid; Clear liquid adds gac 5.3g, stir and be warming up to 80 DEG C, maintaining 1h, suction filtration is removed gac and is obtained the clear liquid that decolours, clear liquid is at 70 DEG C, be evaporated to 63ml, after concentrated solution stirred crystallization 8h, suction filtration obtains crystal, with deionized water wash 2 times, dry 1h at 80 DEG C, obtains 125.0g ALANINE crystal, purity 99.1%.Crystalline mother solution and crystal washing lotion merge 102ml altogether, and ALANINE concentration is 114mg/ml.Yield 91.1%.Transmittance 94.7%
Embodiment 3
Fermented liquid 1.5L, ALANINE concentration 78.7mg/ml, add crystalline mother solution and the crystal washing lotion amalgamation liquid of embodiment 2, obtains fermented liquid clear liquid, use H after crossing 100000 molecular weight ultra-filtration membranes
2sO
4regulate clear liquid pH to 5.0, add FeSO
47H
2o 4.5g, after stirring and dissolving, maintains and stirs, drip the H of 30%
2o
215ml, and be slowly warming up to 50 DEG C, holding temperature and stirring 3h, regulate pH to 7.0 by NaOH solution, use diatomite drainage, suction filtration reaction solution is also washed with water top, obtains clear liquid; Clear liquid adds gac 4.5g, stir and be warming up to 80 DEG C, maintaining 1h, suction filtration is removed gac and is obtained the clear liquid that decolours, clear liquid is at 70 DEG C, be evaporated to 71ml, after concentrated solution stirred crystallization 8h, suction filtration obtains crystal, with deionized water wash 2 times, dry 1h at 80 DEG C, obtains 111.4g ALANINE crystal, purity 99.0%.Crystalline mother solution and crystal washing lotion merge 150ml altogether, and ALANINE concentration is 116mg/ml.Yield 85.0%.Transmittance 96.0%.
Embodiment 1 ~ embodiment 3 amounts to 4.5L fermented liquid, and ALANINE is 354.0g altogether, and obtain ALANINE product 336.7g altogether after treated purifying, average yield is mother liquor 71ml after 94.3%, three crystallizations, and total cycles of concentration is 63.4 times.
Embodiment 4 (one kettle way)
Fermented liquid 1.0L, ALANINE concentration 79.0mg/ml, obtain fermented liquid clear liquid after crossing 50000 molecular weight ultra-filtration membranes, use H
2sO
4regulate clear liquid pH to 5.0, add FeSO
47H
2o 3.0g, after stirring and dissolving, maintains and stirs, drip the H of 30%
2o
210ml, and be slowly warming up to 50 DEG C, holding temperature and stirring 3h, regulate pH to 7.0 by NaOH solution, then add gac 3.0g, stir and be warming up to 80 DEG C, maintain 1h, suction filtration is removed gac and is washed with water top and obtains the clear liquid that decolours, and clear liquid is at 70 DEG C, be evaporated to 48ml, after concentrated solution stirred crystallization 8h, suction filtration obtains crystal, with deionized water wash 2 times, dry 1h at 80 DEG C, obtains 68.7g ALANINE crystal, purity 99.0%.Crystalline mother solution and crystal washing lotion merge 75ml altogether, and ALANINE concentration is 133mg/ml.Yield is 86.1%.Transmittance 98.2%.
Embodiment 5: the process of the mother liquor repeatedly after condensing crystal
Mother liquor (ALANINE concentration is about 97mg/ml, brownish black, thickness) repeatedly after condensing crystal, 1.0L, after diluting 1 times, uses H with deionized water
2sO
4solution regulates pH to 5.0, adds 40gFeSO
4.7H
2o, after it dissolves, drips the H of 30%
2o
2120ml, slowly be warming up to 60 DEG C in the process, holding temperature and stirring 3 hours, pH to 7.0 is regulated by NaOH solution, centrifugally obtain clear liquid, clear liquid adds 10g gac, is warming up to 80 DEG C, holding temperature and stirring 1 hour, suction filtration obtains destainer after removing gac, destainer is evaporated to 300ml at 70 DEG C, after concentrated solution stirred crystallization 8h, centrifugally obtains crystal, 2 times are washed with ALANINE saturated solution, at 80 DEG C, drying 1 hour, obtains 50.4g ALANINE crystal, purity 98.5%.Yield 51.2%.Transmittance 90.1%.
Embodiment 6: the process of the fermented liquid that fermentation is abnormal
The fermented liquid 3.0L (contaminating coccus during the fermentation) that fermentation is abnormal, ALANINE concentration 34.0mg/ml, suction filtration obtains fermented liquid clear liquid after removing thalline, uses H
2sO
4regulate clear liquid pH to 5.0, add FeSO
47H
2o 9.0g, after stirring and dissolving, maintains and stirs, drip the H of 30%
2o
230ml, and be slowly warming up to 50 DEG C, holding temperature and stirring 3h, regulate pH to 7.0 by NaOH solution, use diatomite drainage, suction filtration reaction solution is also washed with water top, obtains clear liquid; Clear liquid adds gac 9.0g, stir and be warming up to 80 DEG C, maintaining 1h, suction filtration is removed gac and is obtained the clear liquid that decolours, clear liquid is at 70 DEG C, be evaporated to 450ml, after concentrated solution stirred crystallization 8h, suction filtration obtains crystal, 2 times are washed with ALANINE saturated solution, dry 1h at 80 DEG C, obtains 54.9g ALANINE crystal, purity 98.6%.Yield 53.1%.Transmittance 90.2%
Embodiment 7
Other is as embodiment 1, only the pH HCl solution of fermentation clear liquid is adjusted to 4.5, obtains 80.8g ALANINE crystal, purity 99.3%.Crystalline mother solution and crystal washing lotion merge 98ml altogether, and ALANINE concentration is 115mg/ml.Yield 68.0%.Transmittance 96.2%.
Embodiment 8
Other is as embodiment 1, and difference is the pH HCl solution of fermentation clear liquid to be adjusted to 6.0, obtains 95.7g ALANINE crystal, purity 98.6%.Crystalline mother solution and crystal washing lotion merge 180ml altogether, and ALANINE concentration is 129mg/ml.Yield 80.0%.Transmittance 93.4%.
Embodiment 9
Other are as embodiment 1, and difference is to add FeSO
47H
2o 3.0g, obtains 95.4g ALANINE crystal, purity 98.6%.Crystalline mother solution and crystal washing lotion merge 170ml altogether, and ALANINE concentration is 119mg/ml.Yield is 79.7%.Transmittance 92.7%.
Embodiment 10
Other are as embodiment 1, and difference is to add FeSO47H
2o 7.5g, obtains 87.8g ALANINE crystal, purity 98.9%.Crystalline mother solution and crystal washing lotion merge 112ml altogether, and ALANINE concentration is 119mg/ml.Yield is 73.6%.Transmittance 97.7%
Embodiment 11
Other are as embodiment 1, and difference is to add 30%H
2o
27.5ml, obtains 94.4g ALANINE crystal, purity 98.5%.Crystalline mother solution and crystal washing lotion merge 205ml altogether, and ALANINE concentration is 121mg/ml.Yield is 78.8%.Transmittance 92.9%.
Embodiment 12
Other are as embodiment 1, and difference is to add 30%H
2o
245ml, obtains 80.3g ALANINE crystal, purity 99.0%.Crystalline mother solution and crystal washing lotion merge 105ml altogether, and ALANINE concentration is 114mg/ml.Yield is 67.4%.Transmittance 97.8%.
Embodiment 13
Other are as embodiment 1, and difference is to add H
2o
2after, be slowly warming up to 30 DEG C, obtain 96.4g ALANINE crystal, purity 98.5%.Crystalline mother solution and crystal washing lotion merge 185ml altogether, and ALANINE concentration is 121mg/ml.Yield is 80.5%.Transmittance 91.5%.
Embodiment 14
Other are as embodiment 1, and difference is to add H
2o
2after, be slowly warming up to 80 DEG C, obtain 91.2g ALANINE crystal, purity 98.8%.Crystalline mother solution and crystal washing lotion merge 99ml altogether, and ALANINE concentration is 121mg/ml.Yield is 76.4%.Transmittance 92.7%.
Embodiment 15
Other are as embodiment 1, and difference is, add H
2o
2and after heating up, holding temperature and stirring 2h, obtain 97.9g ALANINE crystal, purity 98.8%.Crystalline mother solution and crystal washing lotion merge 196ml altogether, and ALANINE concentration is 103mg/ml.Yield is 82.0%.Transmittance 94.3%.
Embodiment 16
Other are as embodiment 1, and difference is to add FeCl
24H
2o 3.2g, obtains 99.8g ALANINE crystal, purity 99.3%.Crystalline mother solution and crystal washing lotion merge 145ml altogether, and ALANINE concentration is 124mg/ml.Yield is 84.0%.Transmittance 96.0%.
Embodiment 17
Other are as embodiment 1, and difference is to add FeNO
31.9g, obtains 99.8g ALANINE crystal, purity 99.4%.Crystalline mother solution and crystal washing lotion merge 152ml altogether, and ALANINE concentration is 118mg/ml.Yield is 84.1%.Transmittance 95.8%.
Claims (12)
1. from ALANINE fermented liquid, extract the method for ALANINE, comprise the steps:
A), after fermentation ends, fermentation liquor solid-liquid separation, obtains the clear liquid containing ALANINE;
B) use acid is by clear liquid pH regulator to 4.5-6.0, adds solvable Fe
2+salt makes Fe
2+the final concentration of ion is 7mmol/L-18mmol/L, adds the H of supernatant volume 0.15%-0.90%
2o
2, be slowly warming up to 40 DEG C-80 DEG C in the process, stir at such a temperature;
C) optionally, regulate pH to 7.0-8.0 with alkali, then solid-liquid separation obtains clear liquid;
D) at 50 DEG C-80 DEG C, add the gac of 0.1-0.3%, stir at such a temperature, remove after gac through solid-liquid separation and obtain destainer, leave standstill after reduction vaporization is concentrated, filter, washing, after drying, obtain ALANINE crystal.
2. the method for claim 1, wherein step a) described in solid-liquid separation process undertaken by adopting ultrafiltration membrance filter fermented liquid.
3. method as claimed in claim 2, the molecular weight cut-off of wherein said ultra-filtration membrane is 50000 dalton to 400000 dalton.
4. the method for claim 1, wherein step b) described in acid be hydrochloric acid or sulfuric acid.
5. the method for claim 1, wherein step b) described in Fe
2+the concentration of ion is 10.8mmol/L.
6. the method for claim 1, wherein step b) described in the pH regulator to 5.0 of clear liquid.
7. the method for claim 1, wherein step b) described in stir continue at least 2 hours.
8. the method for claim 1, wherein step b) described in stir continue at least 3 hours.
9. the method for claim 1, wherein step c) described in alkali be selected from sodium hydroxide.
10. the method for claim 1, wherein steps d) described in stir continue at least 1 hour.
11. the method for claim 1, wherein steps d) described in solid-liquid separation process undertaken by suction filtration.
12. the method for claim 1, are wherein added to recovery in the fermented liquid of next batch by the washing lotion of single batch of crystalline mother solution and washing crystal.
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| CN103601647B (en) * | 2013-11-27 | 2015-06-10 | 南京工业大学 | Method for desalting and decoloring L-alanine fermentation liquor produced by genetically engineered bacteria |
| CN105348122B (en) * | 2015-10-30 | 2017-11-10 | 安徽丰原发酵技术工程研究有限公司 | A kind of purification process of L alanine extreme trace mother liquor |
| CN106520853A (en) * | 2016-12-27 | 2017-03-22 | 秦皇岛华恒生物工程有限公司 | Preprocessing method for L-alanine fermentation liquor produced by means of fermentation method |
| CN113999059B (en) * | 2021-11-15 | 2022-12-13 | 秦皇岛华恒生物工程有限公司 | Amino acid slow release fertilizer and preparation method and application thereof |
| CN114921505B (en) * | 2022-06-28 | 2024-04-16 | 烟台恒源生物股份有限公司 | L-alanine efficient enzymatic conversion and extraction process thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831467A (en) * | 2010-04-21 | 2010-09-15 | 广西大学 | Method for preparing L-alanine by adding laurate alcohol ester phenylacetate |
| CN101974476A (en) * | 2010-08-31 | 2011-02-16 | 安徽华恒生物工程有限公司 | XZ-A26 bacterial strain for producing L-alanine with high yield as well as construction method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831467A (en) * | 2010-04-21 | 2010-09-15 | 广西大学 | Method for preparing L-alanine by adding laurate alcohol ester phenylacetate |
| CN101974476A (en) * | 2010-08-31 | 2011-02-16 | 安徽华恒生物工程有限公司 | XZ-A26 bacterial strain for producing L-alanine with high yield as well as construction method and application thereof |
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| Title |
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